The M.ADP.Pi state is required for helical order in the thick filaments of skeletal muscle.

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.

Kinetics of nucleoside triphosphate cleavage and phosphate release steps by associated rabbit skeletal actomyosin, measured using a novel fluorescent probe for phosphate.

We have measured the kinetics of inorganic phosphate (Pi) release during a single turnover of actomyosin nucleoside triphosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triphosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin-NTP and myosin-NDP-Pi. The steady state intermediates were then mixed with actin. The kinetics of Pi release were measured using a fluorescent probe for Pi, based on a phosphate binding protein [Brune et al. (1994) Biochemistry 33, 8262-8271]. These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATP hydrolysis was followed by chemical quenching of the reaction mix at various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of Pi release are biphasic. The fast phase corresponds to Pi release from the associated actomyosin-ADP-Pi complex. The slow phase measures the rate of the cleavage step on associated actomyosin. At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the Pi burst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin-ADP-Pi formed during the first mix. For ATP at 20 degrees C the rate of the Pi release step is 75 (+/-5) s-1, 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of Pi release varies only slightly with nucleoside structure. The rate constant of the slow phase of the Pi release (measuring cleavage) is highly dependent upon the structure of the NTP substrate.

Electron cryomicroscopy of acto-myosin-S1 during steady-state ATP hydrolysis.

The structure of the complex of actin and myosin subfragment-1 (S1) during steady-state ATP hydrolysis has been examined by electron microscopy. This complex is normally dissociated by ATP in vitro but was stabilized here by low ionic strength. Optimal conditions for attachment were established by light-scattering experiments that showed that approximately 70% of S1 could be bound in the presence of ATP. Micrographs of the unstained complex in vitreous water suggest that S1 attaches to actin in a variety of configurations in ATP; this contrasts with the single attached configuration seen in the presence of ADP. The data are therefore compatible with the idea that a change in attached configuration of the myosin cross-bridge is the origin of muscle force. In control experiments where ATP was allowed to hydrolyze completely the binding of the S1 seemed cooperative.

Kinetics of binding and hydrolysis of a series of nucleoside triphosphates by actomyosin-S1. Relationship between solution rate constants and properties of muscle fibers.

We have measured the steady state kinetics of hydrolysis and presteady state kinetics of binding of the nucleoside triphosphate GTP, CTP, aza-ATP (1-N6-etheno-2-aza-ATP), and ATP by rabbit skeletal actomyosin-S1. The maximum rates of steady state hydrolysis at 10 degrees C at low ionic strength are: CTP, 1.9 s-1 > ATP, 1.3 s-1 > aza-ATP, 0.19 s-1 > GTP, 0.03 s-1. A similar dependence of the rate of steady state hydrolysis upon nucleotide structure has been observed in isometrically contracting muscle fibers in the accompanying paper (Pate, E., Franks-Skiba, K., White, H., and Cooke, R. (1993) J. Biol. Chem. 268, 10046-10053) which strongly suggests that the same biochemical step that limits the maximum rate of hydrolysis of nucleoside triphosphates by actomyosin-S1 in solution also limits the rate of hydrolysis by isometrically contracting muscle fibers. The apparent second order rate constants for the dissociation of actomyosin-S1 by nucleoside triphosphates at 10 degrees C are: ATP, 2.7 x 10(6) M-1 s-1 > aza-ATP, 3.4 x 10(5) M-1 s-1 > GTP, 2.5 x 10(5) M-1 s-1 > CTP, 1.4 x 10(5) M-1 s-1. There is an excellent correlation between the second order rate constant for the dissociation of actomyosin-S1 in solution and the dependence of shortening velocity in glycerinated muscle fibers upon the concentration for ATP, aza-ATP, and CTP (as per accompanying article; Pate et al., 1993). We have used the second order rate constants obtained in solution for the dissociation of actomyosin-S1 by these nucleotides and shortening velocity data obtained with the same nucleoside triphosphates in glycerinated psoas fibers in the accompanying article (Pate et al., 1993) to determine the average distance over which cross-bridges remain attached during unloaded shortening to be 5-12 nm.

Sporotrichosis.

Sporotrichosis is a chronic fungal infection that most commonly involves the skin and lymphatics. It is classified into five forms: classic lymphocutaneous, fixed cutaneous, disseminated cutaneous, primary pulmonary, and systemic sporotrichosis. Its diagnosis is established by fungal culture or by direct immunofluorescence. Safe effective therapy for cutaneous sporotrichosis exists in the form of oral potassium iodide and local heat therapy. However, itraconazole, one of the newer triazole antimycotic agents, appears quite effective against Sporothrix schenckii and may provide a means of reducing both the length of therapy and the relapse rate. Systemic sporotrichosis, although still rare, is becoming more common, especially in immunosuppressed patients. Because of multisystem involvement, both diagnosis and management of patients with systemic sporotrichosis are difficult. Serologic antibody tests such as the slide latex agglutination test are useful to confirm the diagnosis and to determine the effectiveness of antifungal therapy. Intravenous amphotericin B is still considered the drug of choice for systemic sporotrichosis, but the search for a less toxic agent continues. Also, combination drug therapy and surgical intervention are necessary for some patients with extracutaneous sporotrichosis.

The regional graduate medical education program of the University of Washington.

To address problems of geographic and specialty distribution of physicians in the states of Washington, Alaska, Montana, and Idaho, the University of Washington School of Medicine and its affiliated hospitals developed a regional program of graduate medical education beginning in 1971. The program in 1986-87 had 77 percent of the residency positions in the region and involved more than 30 hospitals and clinics. In 1986-87, 58 percent of the residents completing training were in family medicine, internal medicine, or pediatrics. The program is centrally coordinated by the school's associate dean for clinical affairs, who works with departmental program directors and hospital and university administrators.

Intellectual realism: implications for investigations of perceptual perspective taking in young children.

To examine the hypothesis that children's difficulties on traditional perspective-taking tasks are in part due to intellectual realism (inappropriately including what is known to exist in a representation of what is seen), 60 3-, 4-, and 5-year-old children were asked to select representations of various arrangements of blocks. In "visible" arrays, all blocks were visible to subjects; in "hidden" arrays, some blocks were occluded from view. Arrays were presented as already complete ("finished") or were constructed in front of the child ("unfinished"). Consistent with the proposed role of intellectual realism, children performed virtually perfectly on the visually simple (hidden) arrays when presented in finished form but erred on these same arrays when first presented in the unfinished condition. On visible arrays in which what is known to exist is fully congruent with what is actually seen, no difference between finished and unfinished conditions was found. These findings suggest that intellectual realism does influence performance on traditional perspective-taking tasks.

Efficacy of halcinonide cream, 0.1 percent, in the treatment of moderate and severe dermatoses.

The efficacy of halcinonide cream, 0.1 percent, was evaluated in 101 patients with moderate or severe dermatoses. Conditions of these patients included contact dermatitis, atopic dermatitis, nummular eczema, neurodermatitis, stasis dermatitis, dyshidrosis, and various combinations of these disorders. Halcinonide cream was prescribed twice or three times a day for three weeks, and patients were followed-up weekly during this period. Sixteen patients stopped treatment after two weeks because their lesions had cleared. By the end of three weeks, the condition in 46 of the patients had completely resolved, the condition in 39 showed marked improvement, the condition in 10 had improved moderately, the condition in 5 showed mild improvement, and the condition in 1 did not show any improvement. No local or systemic side effects were reported. We conclude that halcinonide cream is an effective and safe topical therapy in the short-term treatment of a variety of moderate to severe steroid-responsive dermatoses.

Topical erythromycin solution in acne. Results of a multiclinic trial.

In a multiclinic double-blind trial, 253 patients with moderate to severe acne vulgaris were treated with erythromycin, 1.5% topical solution (n = 127), or the vehicle (n = 126). The preparations were applied twice daily for 12 weeks. The response to treatment was evaluated by lesion counts and overall clinical judgment at 2, 4, 8, 10, and 12 weeks after initiation of treatment. The reduction in the number of inflammatory lesions, papules, and pustules was significantly greater (p less than 0.01) in the erythromycin-treated group. The global evaluation of the clinical response correlated well with the reduction in the lesion counts. No serious adverse effects were encountered.

Treatment of acne with 5% benzoyl peroxide gel or 0.05% retinoic acid cream.

In a parallel group comparison of a 5% benzoyl peroxide gel with a 0.05% retinoic acid cream, in moderate facial acne vulgaris, significantly more patients in the benzoyl peroxide group experienced overall excellent results than did those in the other group. Both medications, however, were effective in reducing the numbers of both inflammatory and noninflammatory lesions. The benzoyl peroxide gel appeared to produce a more rapid effect on inflammatory lesions than did retinoic acid, and produced significantly less peeling.